Chromatin dynamics regulate diverse nuclear processes that influence cellular viability and tumorigenesis. Different histone modifications are linked to discrete chromatin states and are thought to regulate the extent of accessibility of DNA to transacting factors. One of the most common marks associated with euchromatic genomic regions is methylation of histone H3 at lysine 4 (H3K4). H3K4 can be mono-, di- or tri-methylated, with the tri-methylated species (H3K4Me3) associated with active genes. Inhibitor of growth 3, 4 and 5 (ING3, ING4, and ING5) are tumor suppressors that play a major role in the regulation of cell growth, proliferation, stress responses and aging. Recently, our group found that the PHD fingers of ING proteins recognize methylated H3K4. Furthermore, while ING4 and ING5 interact preferentially with H3K4Me3, the PHD finger of ING3 appears to bind more strongly to the H3K4Me2 histone tail. However, it is unknown how these very similar PHD binding domains differentiate between the Me2 and Me3 histone marks. The objective of this proposal is to determine how binding of PHD fingers targets INGS, ING4 and INGS to the H3K4me2 and H3K4me3 histone tails by characterizing the binding sites of these PHD domains using structural and functional techniques. This will allow us to compare specific interactions of the ING3, ING4 and ING5 PHD fingers with histone peptides at the atomic level. To investigate the molecular basis for the recognition of the Me2 and Me3 histone marks by the PHD finger domains we will co-crystallize ING4 and ING5 with the H3K4Me3 peptide, and ING3 with both the H3K4Me3 and H3K4Me2 peptides. Once we have analyzed the structural data we will use this information to make specific mutations in the PHD domains of ING3, ING4 and INGS to test which residues are most important for the interactions with the Me2 and Me3 peptides. We will measure the effect of these mutations on peptide binding using NMR and tryptophan fluorescence by titrating in histone peptides to determine their dissociation constants (Kds). The functional significance of these mutations will also be tested in vivo by chromatin immunoprecipitation and fluorescence microscopy. These data will provide critical structural insights into the molecular mechanism used to discriminate between very similar histone marks by the PHD domains of ING3, ING4 and INGS, and impart a greater understanding of the mechanistic basis required to effectively read the histone code. ING3, ING4 and INGS are tumor suppressor proteins involved in controlling the way DNA is packaged within our cells. Understanding how higher order DNA structures effect in gene expression is important as loss of this regulation may underlie many disease processes, particularly cancer.